PROJECT SUMMARY Heme-containing proteins are among the most abundant metalloproteins in nature. A significant subset of these catalysts perform reactions utilizing dioxygen (O2), including cytochrome c oxidase (CcO) which reduces dioxygen to water, and cytochrome P450 (CYP) which activates dioxygen in order to oxidize organic substrates. The functions of CYP and CcO rely on their abilities to reduce O2 to water. In an ?uncoupled? process, some equivalents of reductant are wasted and reactive oxygen species (ROS) such as H2O2 are released. ROS are known to lead to oxidative stress and a variety of diseases in the human body, so developing an understanding of this uncoupling is important. It is hypothesized that a catalytic Fe?OOH intermediate is the site of bifurcation between H2O and H2O2 formation. This hydroperoxy intermediate is ubiquitous in heme-containing enzymes including cytochrome c peroxidase, heme oxygenase, and prostaglandin H synthase. In all cases, proper proton delivery is necessary for O?O bond cleavage. Mutagenesis studies of P450cam have made it clear that the role of protons and H-bonding are important in understanding selectivity, but there is debate as to how the conserved residues prevent uncoupling. Additionally, the canonical mechanism involves one proton addition to either the proximal or distal oxygen atom of the Fe?OOH intermediate to yield H2O2 or H2O, respectively, but there is no direct evidence of this stoichiometry. Our preliminary data suggests that the desired distal protonation may involve a higher dependence on protons. Therefore our goal is to study what governs the selectivity between formation of H2O or release of H2O2 from this critical Fe?OOH intermediate. Our work in this proposal seeks to understand the selectivity of H2O and H2O2 formation from heme enzymes utilizing a simple model system. Synthetic analogues provide the advantage of allowing us to systematically vary and control structural entities, enter the catalytic cycle in new places, and independently synthesize intermediates. Therefore, we propose to study the selectivity of O2 activation by Fe-porphyrin catalysts proceeding through the same Fe?OOH intermediate. First, we will explore how various reaction conditions (concentration, pKa, and structure of the acid) affect selectivity in the catalytic oxygen reduction reaction (ORR). Secondly, we will study a variety of catalysts with varied H-bonding motifs to better understand how the residues in an active site may influence selectivity. Additionally, we will explore the non-catalytic reactivity of the Fe?OOH intermediate to gain independent measures of the relative rates of H2O and H2O2 formation under varied conditions. Ultimately, our goal is to understand how the reaction conditions and H- bonding networks affect H2O versus H2O2 selectivity in a model system. This understanding will provide insight into how enzymes can control the reactivity of the critical Fe-hydroperoxy intermediate to minimize ROS formation in a variety of heme-containing active sites.